Synthetic transfection vectors

ABSTRACT

An inorganic particle, to which is bonded a cell binding component and a nucleic acid, is provided for delivery of a nucleic acid to a cell. The disclosed particle acts as a synthetic vector for achieving efficient transfection of associated nucleic acid into a cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/702,381, filed Oct. 31, 2000, now abandoned which is continuation ofU.S. application Ser. No. 09/062,145, filed Apr. 17, 1998, now U.S. Pat.No. 6,153,598, which is a continuation of U.S. application Ser. No.08/583,195, filed Jan. 4, 1996, now abandoned, which is a file wrappercontinuation of U.S. application Ser. No. 08/211,041, filed Mar. 16,1994, now abandoned, which is a U.S. national stage application filedfrom International patent application No. PCT/GB92/01703, filed Sep. 16,1992.

FIELD OF THE INVENTION

This invention relates to synthetic transfection vectors.

This invention is in the field of gene therapy and concerns the designand use of an entirely novel means of safely introducing therapeuticgenes into mammalian and human cells to achieve various useful effects.

Steady progress over the past twenty years in the field of geneticengineering, nucleic acids research, chromosome mapping, DNA cloning,and other related fields has brought modern medicine to the threshold ofgene therapy. It may be possible to treat some diseases by constructingpieces of DNA or RNA which code information which could correct thephysiological malfunction causing the disease. It may also be possiblein agriculture to make certain beneficial changes in the protein productof some animals or plants.

A crucial limiting factor in progress in this field is the difficulty ofcausing such new genes to enter the cells of intact organisms where theycan commence doing their work. For some small animals and plants, geneshave been introduced into the small number of cells involved in theearly embryo and so caused to replicate and ultimately appear in many orall of the cells of the adult organism. However, this approach is notviable for treating diseases in human children or adults where thedisease is discovered after conception or more commonly after birth.Further, in agriculture, this method proves to be extremely expensiveand difficult to carry out when large numbers of large animals such ascattle are meant to be so treated.

A variety of methods have been attempted for introducing genes intoadult animals. These methods include direct injection of naked DNAplasmids into individual cells, attempts at reapplying calcium phosphatetransfection techniques, inclusion of DNA into liposomes, andconstruction of simulated viruses which can carry the new DNA as a sortof infection. The greatest progress has been made with a group oftechniques in which DNA is coated onto gold colloid particles and theparticles then subject to powerful electromagnetic fields in order toaccelerate them to high speeds and so to hurl them against the cellwalls of tissues. The particles plunge through the tissue surface andmany viable DNA chains arrive inside the cell along with theirnon-degradable gold carrier. To reach tissues other than skin, asurgical operation is performed, and e.g. the tip of the liver isexposed and then a bombardment is carried out. This permits access onlyto surface layers of exposed tissues, is obviously injurious (sincepetechial hemorrhages immediately appear on the tissue surface), anddeposits substantial amounts of non-degradable degradable gold in thetissues.

In the method of this invention, the new DNA, RNA, plasmids, ribosomalparticles, nucleic acid binding proteins and any other necessarymolecules are caused to adhere to the outer surface of any one of avariety of metal oxide or mixed metal crystals of coated or uncoatedtype or to He attached to the surface of or included in the body of avariety of other types of biodegradable particles of appropriate sizeand capable of surface attachment to a cell adhesion molecule. Theseparticles are in the size range of 5 to 100 nm in diameter including allattached coatings and other surface molecules. Included on the surfaceis one of a variety of nerve adhesion molecules or muscle adhesionmolecules which bind to the surface of nerve and muscle cells, butpreferrably to muscle cells.

When such particles are constructed and then administered by routinepercutaneous intramuscular injection, an exceedingly safe and efficienttransfection process is initiated. The particles adhere to the outersurfaces of muscle cells and to the outer surfaces of the axon terminiof motor nerve cells or preferably to the dendritic or sensory processof sensory axons within the muscle. After adherence, the particles areingested into the nerve and muscle cells by a natural process termedadsorptive endocytosis.

Experiments carried out by the inventor have demonstrated a surprisingefficiency for the uptake of such particles after intramuscularinjection. Further, particularly when such particles are made of ironsalts, the particles are completely biodegradable. Normally, particulatematerial injected into muscle is rapidly cleared by the lymphatic systemand the particles are taken into lysosomal vesicles where they aresubject immediately to degradative enzymes. However, the inventor hasshown that when the process of adsorptive endocytosis by muscle cells isentrained, the bulk of the injected material is carried into protectedcompartments within neural and muscular cells.

Many cells have means of destroying any foreign DNA or RNA which appearsin their cytoplasmic compartments, however muscle cells are uniquelyineffective at destroying incoming nucleic acids. In this fashion, andusing intramuscular injection, the agents can be caused to enter thevery large intracellular volume provided by the cells of muscles. Uponuptake by neurons, it is also possible to take advantage of the naturalability of the dendritic processes of neurons to carry out proteinsynthesis from RNA at great distance from the controlling influence ofthe neuron cell body. Use of sensory specific nerve adhesion moleculessuch as Nerve Growth Factor is helpful at efficiently selecting sensoryrather than motor neurons where this is useful. In some situations, itmay be useful to inject the agent into or near and dorsal root ganglionso that the agent can be carried by axonal transport to reach all of thetissues innervated by sensory processes from cells in the ganglion.

Treatment of muscle cells or treatments where gene therapy products aredumped into the neuromuscular synapse after production in the nerveprocess terminus are particularly helpful for treating disorders such asmuscular dystrophy or other diseases which particularly affect muscle orfor treating diseases which affect neuromuscular transmission.

It must be noted, however, that such agents are sufficiently small thatthey can be safely injected intravenously. Because of their potentiallyhydrophilic coatings with e.g. dextran, the inventor has shown extendedplasma half life for such agents with up to 25% of the initial injectateremaining in circulation for over four 10 hours. This provides targetingaccess to a wide variety of cells in the blood marrow, circulatingblood, and various glands and tissues. In all these cases, selection ofappropriate targeting molecules for these particles will causepreferential adsorption to various useful cell types. While efficiencyof phagocytosis of selectively adsorbed particles varies among tissues,there are a very wide variety of accessible intracellular sites. Whenthe metal oxide core is constructed in such a way as to demonstratesuperparamagnetism, then external magnetic fields (as from U.S. Pat. No4,869,247) can be used to aid in targeting the agents.

In one example of synthesis of such compounds, the nucleic acidattachment to the particle is effected by specific nucleic acid bindingproteins. A DNA plasmid or strand is constructed to include both thedesired treatment gene and a segment with very high affinity for aselected nucleic acid binding the attachment DNA segment to immobilelatex particles using a cyanogen bromide immobilisation technique.Various nucleic acid binding proteins and other cell constituents arethen passed through an affinity column made up to such DNA tagged latexparticles. The specific fraction of nucleic acid binding protein is theeluted for use in making the particle.

A mixture of ferrous and ferric chloride salts is dissolved in asaturated dextran solution after the fashion of U.S. Pat. No. 4,452,773and precipitated by addition or 7.55 ammonia solution. The product isthen moved into 0.1 M acetate buffer pH 6.4 by Sephadex 150 columnfiltration, concentrated with Amicon Centriprep 30 ultrafilters, andpassed through a 2.5 cm by 20 cm column of Sephacryl 200 to cleargelatinous hydrous oxides and excess dextran.

The particles are then filtered at 200 nm and 100 nm and next gentlyoxidised in 20 mM NaIO₄. The NaIO₄ is cleared with PD-10 sephadexcolumns and the same column used to transfer the particles into a pH 8.0borate buffer solution. The nerve adhesion molecule such as wheat germagglutinin or transferrin or nerve growth factor with appropriateblocking of active sites (Ca and Mn chlorides) and the appropriatenucleic acid binding protein with small nucleic acid fragments to blockthe active site are then incubated with the particles for 8 to 12 hours.After this, remaining active sites are blocked by adding 1 M glycine for2 additional hours, and the mixture then reduced with NaBH₄ for onehour. After reducing the covalent bonds, the particles are moved intoHEPES 20 mM pH 7.4 buffer through PD-10 columns which also serve toclear unreacted glycine, NaBH₄, and any dissolved iron salts. Theproduct is diluted in HEPES buffer, then reconcentrated with AmiconCentriprep 100 ultrafilters to help clear unbound proteins, and thenpassed through Sephacryl 200 or other Sephacryl size column to clearadditional unreacted protein.

The output from these columns is then reconcentrated with AmiconCentriprep-100 ultrafilters and subject to two rounds of affinitypurification. The first round is on a column carrying the muscle surfaceor neural surface or other desired cell surface marker. In this fashion,all particles which will not adhere to the desired target cells areeliminated. The affinity fraction is eluted, diluted, reconcentrated,and the subjected to a second affinity purification but this timeagainst a column with immobilised DNA fragments which are recognised bythe nucleic acid binding protein now ligated to the particle surface.

The highly purified product of the second affinity step is now dilutedin HEPES 20 mM pH 7.4, reconcentrated with Amicon Centriprep-100 orsimilar ultrafilters and then exposed to the genetic material to bedelivered. When a mixed plasmid or strand is used, the binding proteininteracts with the binding portion of the DNA and the large nucleic acidmolecule carries with it the active gene of interest. It is alsopossible to use nucleic acid binding proteins which bind directly to agene or segment of RNA or DNA of interest when such binding proteins areavailable.

The particles with bound DNA are passed through a Sephacryl column toclear unbound nucleic acid if desired and are now ready forconcentration and dilution in an appropriate physiological solution forintramuscular injection. The agent is now injected into muscle whereuponnatural processes of adhesion and endocytosis complete the genetransfection into the selected cell type.

In another example of the preparation, the initial precipitation of theiron salts is done by dropwise addition to ammonia solution without thepresence of any coating dextran or other molecule. The resultingsuspension is spun in a centrifuge at 500 g for 10 minutes and thepellet washed and resuspended in distilled water and the process thenrecreated but with a wash with 0.01N HCl. The resulting stable colloidis then exposed to a mixture of adhesion molecule protein, nucleic acidstrands and/or nucleic acid binding proteins. After an incubation withgentle non-magnetic stirring one hour, the remaining reactive sites onthe particles are blocked by the addition of dextran or albumin,protein. The particles are then passed through sephadex 150 andSephacryl 200 columns then affinity purified by means of the celladhesion molecule using for instance a column of affinity labelledagarose, sepharose, or latex beads.

In yet another example of the preparation, the initial precipitation iscarried cut by preparing a solution of very strong buffer such as 1Molar or higher concentration of HEPES or Tris at a pH of 7.4. Thenucleic acids, any desired dextran, and or targeting proteins andnucleic acid binding proteins are added directly to this initial strongbuffer. The mixture of dissolved ferrous and ferric iron salts itaqueous solution or in a solution containing dextran and/or proteinand/or nucleic acids is then added dropwise to the buffer solution. Inthis fashion, the particles are formed in a rigidly buffered solutionand so many fragile protein and peptide molecules can be used to formthe particle coat where such molecules are necessary for targeting, forintroducing ribonucleoprotein or ribasomal protein or other aspects oftranscription signalling or actual transcription mechanism proteinsalong with the DNA or RNA. The product of this precipitation reaction isthen further blocked with dextran or albumin if necessary, then purifiedwith sephadex 150, sephacryl 200, Amicon ultrafilters and a affinitycolumns as described above.

In yet another version of the synthesis, there is no nucleic acidbinding protein used but only a cell surface adhesion molecule. Insteadof the nucleic acid binding protein, a complementary fragment of thenucleic acid of interest is bound to the particles by a cyanogen bromideor other type of binding reaction or by adherence to an uncoatedparticle type. The gene or interest is then attached to the particle byits interaction with the bound complementary fragment after whichpurification steps are carried out as described above.

In summary, the present invention provides a synthetic transfectionagent, the corresponding vector without the nucleic acid, and anycombination of the components thereof. It will be appreciated that thesynthetic transfection agent is based on precipitation of one of avariety of ceramic metal oxide particles similar in size to a virus. Themetal oxide particle is coated with dextran or otherbiologically-tolerable polymer during the precipitation process.Chemically, the basic structure is similar to drugs in current use asmagnetic resonance contrast agents.

The dextran or other coating of the particle is used as a framework towhich various other types of molecules are then covalently bound.Typically, a targeting molecule such as an antibody or antibodyfragment, or some other useful cell adhesion molecule is used. Thiscauses the particle to adhere selectively to certain desirable celltypes, e.g. a gp120 fragment to promote adherence to CD4 positive cells.In addition to the targeting molecule, it is also possible to attach anucleic acid binding protein or short cDNA sequence to the dextran coat.In this fashion, particles can be produced with appropriate nucleic acidbinding proteins and targeting molecules, and then subsequently loadedwith the therapeutic DNA.

For intravascular administration, the particle size determines serumhalf-life and destination. Larger particles tend to be cleared into thereticuloendothelial cells by phagocytosis, while small particles achievedestination determined more completely by their targeting molecule.

These particles can also be administered intramuscularly where they cangain entry into muscle cells and also can be ingested by nerve terminalsin the muscle and subsequently subjected to axonal transport from theperiphery towards the neural cell bodies in the central nervous system.In this fashion, mimicking the route of the Herpes virus, anintramuscular infection can be used to deliver DNA across theblood-brain barrier for therapeutic purposes in selected regions of thenervous system. The axonal transport route also provides access toSchwann cells which line the axons.

It is further possible to provide the particles in aerosol form forpulmonary administration. A variety of other routes of administrationare also feasible, including intravenous administration.

The particulate carrier is well suited for treating diseases involvingthe reticuloendothelial system through intravascular and inhalationalroutes, and to treat GI mucosal cells by enteral routes, as well as forintramuscular injection for access to muscle cells. Access via theintraneural route, to CNS and ganglion cells, is provided byintramuscular and intradermal injection.

It has been demonstrated that the particles are ingested by humanmacrophages, T-cells and osteogentic sarcoma cells, and that there isslow clearance of the particles from the blood stream in a rabbit, with25% of the injected dose remaining in the circulation after four hours.Particles have coated with dextran and conjugated to both anti-CD4 andDNA polymerase as a nucleic acid-binding protein, with subsequent exposeto and uptake of DNA plasmids onto the particle surface. Particles mayalso be coated with DNA directly, rather than with dextran.

The particles are biodegradable in the sense that they can break sown,in vivo, to materials that are essentially harmless. Thus, for example,while foreign materials such as gold particles may be found intact incells years later, iron oxide particles dissolve readily into oxygen andiron, both of which are of course naturally present in abundance incells and which then participate in normal cellular metabolism, storageand reuse. Iron poses some risk of toxicity when present in highamounts. The potential toxicity of ferrites is reduced by ensuring thatthey dissolve slowly, at a rate no faster than the cells' ability toprocess the elemental iron. Extension of the degradation rate for theparticles can be achieved by ensuring that the original preparation isfree of hydrous oxides of iron that are similar in overall size andchemical composition to ferrites but dissolve very rapidly.Additionally, by doping in other elements such as palladium (which iswater-soluble in elemental form unlike gold) which can improve thestability of the ferrite, the rate of breakdown can be slowed.

The ceramic particle-based system disclosed here has advantages overviral vectors, in that there is no risk of infection or of introductionof unwanted viral genes. It has advantages over the gold colloid systemin that the particles are biodegradable. Further, since they may have acoat to which proteins may be covalently bound, these particles can beinjected into the body via less drastic and more traditionalpharmaceutical routes. The facility with which various proteins andenzymes can be bound to the particle surface makes them far moreflexible and complex as delivery agents than simple lipid spheres.

Because of the size of the particles as used in this invention, they canbe filter-sterilised and subjected to affinity chromatography before DNAis loaded. This means that particle carriers can be sold independent ofthe DNA and can serve as a convenient synthetic vector for a widevariety of applications. It is also convenient to label the metal oxidecore with radioactive emitters where this is useful to trace theirdistribution.

The preparation of inorganic, metal oxide particles for therapeuticdelivery is described in WO-A-9204916. In the illustrative Example thatfollows, the preparation of the particles gives desirable homogeneityand avoidance of water-soluble materials that may adversely affect thedesired slow metabolism. The Example obviates column chromatography, forwhich the use of centrifugal concentrators has been substituted, andinvolves the corresponding omission of NaCl elution buffers. EDTA isused as a chelating agent in the buffer used in the first washing steps.This apparently dissolves the iron in the hydrous oxides, but does notdissolve the well-formed ferrites. The result is a stable and uniformparticle preparation with low toxicity (because it is a substantiallypure ceramic preparation).

EXAMPLE

Use double distilled water (not de-ionised) to make up the reactionmixture. The following steps are conducted:

Add 1.5 ml of 33% NH₃ to 4.5 ml of hot dH₂O (to make up 7.5% NH₄OH) andleave standing in a capped universal tube in the water bath and bring to60° C.

Dissolve 1.25 g Dextran (MW 10,000) in 2.0 ml of ddm₂O then dissolve 225mg FeCl₃.6H₂O in the dextran solution. Alternatively, a trivalentlanthanide chloride may be substituted for 10 to 50% of the FeCl₃. Whenthis is done, the subsequent post-reaction incubation is extended to twohours.

Dissolve 100 mg FeCl₂.4H₂O in the Fe₃/dextran solution then place themixture in a 60° C. water bath for two minutes before starting togradually add 5 ml of hot 7.5% NH₃ solution (60° C.). The product isleft to stand in the 60° C. water bath for fifteen minutes.

The reaction product (dextran-coated ferrites) is spun at 1,000 g for 10minutes and any precipitate is discarded. This process is repeated tocomplete three spins and the supernatant then applied to PD-10 columnsequilibrated with 0.1 M NaAcetate buffer, pH 6.8 with 5 mM EDTA.

The black eluted fraction is diluted 1:3 with EDTA/Acetate buffer thenconcentrated to one-tenth the initial volume with Amicon Centriprep-100ultrafilters. The retentate is then diluted 1:10 with EDTA/Acetatebuffer then concentrated to a volume of 1.5 ml with the C-100ultrafilters.

Add 0.30 ml of 20 mM NaTO₄ to the dextran ferrite solution (approx. 1.5ml) while stirring then gently tumble or sake for 60 minutes at roomtemperature in the dark.

At the end of the 60-minute periodate incubation, the reaction isterminated by applying the reaction mixture to the PD-10 columnsequilibrated with 20 mM borate buffer (pH 8.5).

An active site blocking solution is prepared using 100 mM MnCl₂/CaCl₂for WGA binding reactions. Alternatively, e.g. calf thymus DNA can beused where the protein active site to be protected is on a nucleic acidbinding protein.

Dissolve 10 mg of the protein (e.g. DNAse free DNA pol 1, Klenowfragment, integrase, useful proteins for subsequent translation steps,nucleic acid packaging protein and anti-CD4, WGA, or othercell-targeting protein) in 500 μl of 20 mM Na borate buffer, pH 8.5 atroom temperature. The protein solution can be diluted to 12 ml withborate buffer, then concentrated with Centriprep-10 concentrators toremove DTT, glycerol, NaN₃ and other undesirable storage additives.

Add 10 μl of the blocking solution to the protein/borate solution thenmix 2.0 ml of oxidised magnetite dextran with 500 μl of theprotein/borate solution. Pipette 20 μl of the blocking solution into the2.5 ml protein-dextran-magnetite mixture and mix well, then incubate for6 to 18 hours at room temperature in a gentle tumbling or shakingdevice.

After the incubation, add 100 μl of 0.5 M glycine to the reactionmixture and incubate an additional 2 hours. Then add 250 μl of 0.25 MNaBH to the magnetite-dextran-protein solution and allow to stand for 60minutes, shaking periodically to release H₂ gas. At the end of theincubation, pass the reaction mixture through PD-10 columns equilibratedwith 20 mM HEPES buffer, pH 7.4. Dilute the eluant 1:5 with HEPES bufferthen concentrate with Centriprep-100 ultrafilters.

An affinity purification step is optional and detail is given for usewith a WGA(lectin) targeting protein. Apply final retentate to affinitycollins (20 mM HEPES), wash with HEPES, then carry out specific elutionwith 1 M NAcGlu in HEPES buffer, pH 7.4. Pass the specific eluantthrough PD-10 columns equilibrated with HEPES to remove NAcGlu, Mn andCa.

The desalted output is then diluted to a volume of 24 ml with HEPESbuffer and concentrated with Centriprep-100 concentrators. The finalretentate is sterilised by spinning at 500 h for one hour in 0.22 μmcentrifugal microfilters.

The purified, sterilised synthetic vector particles 10 can now be storedat 4° C. for use within one to two weeks. They should not be frozen orlyophilised.

DNA adhesion with the DNA of interest can be done immediately prior tothe transfection. The particle solutions are incubated with the DNA ofinterest with gentle tumbling or shaking for 6 to 24 hours.

Depending on the experimental or therapeutic protocol, the DNA-loadedvector solution may then be applied to cell cultures at a concentrationof 1 mg/ml (approx. 5 mM Fe) of the synthetic vector (the final productof the preparation is 25 to 50 mg of synthetic vector). To assessefficiency, it may be compared to unadsorbed DNA solution.Alternatively, the DNA-loaded synthetic vector may be administered by IVor IM routes for in vivo use at 10 to 100 mM concentration.Non-preciptating magnetic-based separation techniques can be used toseparate unbound DNA from particles. Where smaller DNA molecules areused, the separation can be done with Centriprep-100 concentrators.

We claim:
 1. A gene delivery vector comprising a ferrite particle havinga polymeric coating to which a targeting polypeptide molecule and anucleic acid binding protein are covalently bound, wherein a nucleicacid of interest is bound to said nucleic acid binding protein.
 2. Thegene delivery vector according to claim 1 wherein said targetingpolypeptide molecule is selected from the group consisting of wheat germagglutinin, transferrin and nerve growth factor.
 3. The gene deliveryvector according to claim 1 herein said targeting polypeptide moleculeis an antibody or antibody fragment.
 4. The gene delivery vectoraccording to claim 1 wherein said polymeric coating is a dextrancoating.
 5. The gene delivery vector according to claim 1 wherein saidferrite particles are produced by the preparation of a mixture offerrous and ferric chloride.
 6. An injectable composition comprising thegene delivery vector according to claim 1 and a physiologicallyacceptable diluent.
 7. A ferrite particle having a polymeric coating towhich a targeting polypeptide molecule and a nucleic acid that iscomplementary to a nucleic acid of interest are covalently bound.
 8. Theferrite particle according to claim 7 wherein said targeting polypeptidemolecule is selected from the group consisting of wheat germ agglutinin,transferrin and nerve growth factor.
 9. The ferrite particle accordingto claim 7 wherein said targeting polypeptide molecule is an antibody orantibody fragment.
 10. The ferrite particle according to claim 7 whereinsaid polymeric coating is a dextran coating.
 11. The ferrite particleaccording to claim 7 wherein said ferrite particles are produced by thepreparation of a mixture of ferrous and ferric chloride.
 12. The ferriteparticle according to claim 7 wherein said nucleic acid of interest isbound to said nucleic acid.
 13. An injectable composition comprising theferrite particle according to claim 7 and a physiologically acceptablediluent.